Haemophilia A is an X-linked disorder characterised by the absence or insufficient amount of functional factor VIII, a 330 kD glycoprotein molecule produced by the liver as a single polypeptide chain of 2332 amino acids. This deficiency affects 1 in 10,000 males and can result in uncontrolled bleeding in joints, muscles and soft tissues. Patients affected by the severe form of the disease (FVIII activity lower than 1% of normal level) suffer from spontaneous bleedings. Patients with corresponding FVIII activity from 1 to 5%, or higher than 5% are defined as moderate or mild haemophilia A, respectively, and suffer from limited bleeding occurring after minor trauma or surgery. The coagulation pathway can be restored by administration of FVIII concentrates prepared from plasma or produced by recombinant cDNA technology.
The human FVIII gene has been isolated and expressed in mammalian cells, as reported by various authors, including Wood et al. in Nature (1984) 312: 330-337 and the amino-acid sequence was deduced from cDNA. U.S. Pat. No. 4,965,199 discloses a recombinant DNA method for producing FVIII in mammalian host cells and purification of human FVIII. The human FVIII detailed structure has been extensively investigated. The cDNA nucleotide sequence encoding human FVIII and predicted amino-acid sequence have been disclosed for instance in U.S. Pat. No. 5,663,060. In a FVIII molecule, a domain may be defined as a continuous sequence of amino-acids that is defined by internal amino-acid sequence homology and sites of proteolytic cleavage by a suitable protease such as thrombin. The FVIII proteins has been described to consist of different domains, which for the human amino-acid sequence correspond to: A1, residues 1-372; A2, residues 373-740; B, residues 741-1648; A3, residues 1690-2019; C1, residues 2020-2172; C2, residues 2173-2332. The remaining sequence, residues 1649-1689, is usually referred to as the FVIII light chain activation peptide. FVIII is produced as a single polypeptide chain which, upon processing within the cell, is rapidly cleaved after secretion to form a heterodimer made of a heavy chain containing the A1, A2 and B domains and a light chain made of the A3-C1-C2 domains, according to Kaufman et al. (1988, J Biol Chem 263:6352-6362). The two chains are non-covalently bound by divalent cations. Both the single-chain polypeptide and the heterodimer circulate in plasma as inactive precursors, as taught by Ganz et al. (1988, Eur J Biochem 170:521-528). Activation of factor VIII in plasma initiates by thrombin cleavage between the A2 and B domains, which releases the B domain and results in a heavy chain consisting of the A1 and A2 domains, according to Eaton et al. (1986, Biochemistry 25:505-512). Human recombinant FVIII may be produced by genetic recombination in mammalian cells such as CHO (Chinese Hamster Ovary) cells, BHK (Baby Hamster Kidney) cells or other equivalent cells.
Pratt et al. (1999, Nature 402:439-42) disclose the detailed structure of the carboxy-terminal C2 domain of human FVIII, which contains sites that are essential for its binding to von Willebrand factor (vWF) and to negatively charged phospholipid surfaces. This structure, which reveals a beta-sandwich core from which two beta-turns and a loop display a group of solvent-exposed hydrophobic residues, partly explains mutations in the C2 region that lead to bleeding disorders in haemophilia A. According to Gale et al. (2000, Thromb. Haemost 83:78-85), of the at least 250 missense mutations that cause FVIII deficiency and haemophilia A, 34 are in the C domains.
FVIII is a cofactor of the intrinsic pathway of the coagulation cascade, which acts by increasing the proteolytic activity of activated factor IX over factor X, in the so-called tenase complex formation. Patients suffering from haemophilia A present bleedings which are either spontaneous in the severe form of the disease, or occur after trauma in the mild/moderate forms.
Haemophilia A patients are usually treated by replacement therapy, which consists in infusing human FVIII either purified from pools of donor plasma, or obtained by cDNA recombination technology.
The majority of the patients are immunologically unresponsive to these infusions, but for yet unclear reasons, 25% of them mount an IgG immune response towards FVIII, which can result in complete inhibition of the procoagulant activity of infused FVIII (Briët E et al. in (1994) Throm. Haemost.; 72: 162-164; Ehrenforth S et al. in (1992) Lancet, 339:594). Such specific IgG, which belong to the IgG 1, 2, 4 subclasses, are called FVIII inhibitors. Published studies have demonstrated that the anti-FVIII immune response is polyclonal, and primarily directed towards the A2, A3 and C2 domains (Scandella D et al. in (1989) Blood; 74: 1618-1626; Gilles J G et al. in (1993) Blood; 82: 2452-2461).
Recent studies using human monoclonal antibodies derived from the peripheral memory B cell repertoire of inhibitor patients indicated that important epitopes are also located on the C1 domain (Jacquemin M et al. in (2000) Blood 95:156-163). The mechanism by which anti-FVIII antibodies interfere with the function of FVIII are numerous, including proteolytic cleavage of FVIII and interaction with different partners such as von Willebrand factor (vWF), phospholipids (PL), FIX, FXa or APC. Most of these mechanisms are now well described in studies using mouse or human anti-FVIII antibodies. Thus, antibodies can reduce the rate at which FVIII is activated by either binding to a proteolytic cleavage site or by inducing a 3D conformational change in FVIII that renders it less amenable to proteolysis. Antibodies interfering with the binding of vWF to FVIII appear to be very efficient as inhibitors, as shown in recent studies using human monoclonal antibodies directed towards the C2 domain, which is one of the major vWF binding sites (Jacquemin M et al in (1998) Blood; 92:496-501). Suppressing the production of inhibitors and establishing a state of immune unresponsiveness to FVIII remains a major goal. The medical community is, however, far from reaching these goals, due basically to the limited understanding of the mechanisms underlying specific antibody production and regulation.
Presently, to control such an immune response, several treatments are used including bypassing agents such as desmopressin (DDAVP), agents promoting coagulation such as prothrombin complex concentrates (PCC) or activated PCC, recombinant FVIIa, plasmapheresis and infusions of large or intermediate doses of FVIII (200-300 IU/kg body weight or 25-50 IU/kg body weight, respectively). However, none of these methods are satisfactory and they are all very costly.
Based on these observations and on the understanding of the mechanisms of immune tolerance, anti-idiotypic antibodies appear to be a promising way of treating inhibitors. In fact, it is well established that tolerance to self-protein is first induced at an early stage by clonal deletion of self-reactive B and T cells in the bone marrow and the thymus, respectively. However, not all self-reactive lymphocytes are eliminated by central deletion. Auto-reactive B cells are a common feature of peripheral blood, as well as low- or intermediate-affinity self-reactive T cells. A number of mechanisms by which such auto-reactive cells are rendered non-functioning or are deleted in the periphery have been described. Anti-idiotypic antibodies can represent a third level of tolerance maintenance in this general scheme, with their capacity to fine tune the function of antibodies and maintain a subtle equilibrium between complementary idiotypes expressed on B and T cells.
A good indication of how anti-idiotypic antibodies can exert a regulatory mechanism in the periphery is provided by the demonstration that healthy individuals with normal levels of FVIII produce significant titres of inhibitory antibodies to FVIII (Algiman M et al. in (1992) Proc Natl Acad Sci USA 89:3795-3799; Gilles J G et al in (1994) J Clin Invest 94:1496-505), the activity of which is undetectable in plasma because of the presence of complementary anti-idiotypic antibodies. However, such a FVIII inhibitory activity can be readily detected when anti-FVIII antibodies are purified by a combination of chromatography and specific immunoadsorption over insolubilized FVIII. The FVIII inhibitory capacity of affinity-purified antibodies was demonstrated to be equal to that of anti-FVIII antibodies purified from haemophilia A patient's plasma with high level of inhibitors, as measured by Bethesda assay (Gilles J G et al. in (1994) J Clin Invest 94:1496-505).
Such neutralising anti-idiotypic activity has also been detected in a group of patients successfully desensitised by administration of high doses of FVIII (Gilles J G et al. in (1996) J Clin Invest 97:1382-1388). The study demonstrated that the concentration of anti-FVIII antibodies, purified by the same procedure as for healthy donors, did not change during desensitisation and that antibodies maintained their capacity to inhibit the procoagulant function of FVIII, even though the titration of inhibitor using the Bethesda assay in plasma was reduced to undetectable levels. This pointed to the potentially important function of anti-idiotypic regulation in tolerance to FVIII molecule. Therefore, any novel therapy inducing an increased production of anti-idiotypic antibodies can be of interest in the treatment of patients with FVIII-inhibitors. A first approach along these lines has been reported, in which patients were treated with injections of immune complexes made of FVIII and autologous specific antibodies towards FVIII, which resulted in a significant reduction in the level of circulating FVIII inhibitors, which were neutralised by corresponding anti-idiotypic antibodies (Gilles J G, Arnout J. XXI International Congress of the World Federation of Haemophilia 1994 April; abstract). Such an approach can open the way towards new therapeutic strategies, which target FVIII inhibitors at potentially low cost compared to presently available treatments.
Previous findings from the inventors' laboratory have shown that anti-idiotypic antibodies exert physiological properties in the homeostasis of the anti-FVIII immune response. Thus, the peripheral blood of healthy individuals contains antibodies specific for FVIII; some of which have the property of inhibiting the procoagulant function of FVIII (Gilles JGG & Saint-Remy JMR in (1994) J Clin Invest 94: 1496-1505). In these individuals, the function of FVIII is actually not altered, as antibody-mediated FVIII inhibition is neutralised by specific anti-idiotypic antibodies. We therefore concluded that anti-idiotypic antibodies have a physiological relevance in the maintenance of normal FVIII activity.
Moreover, the inventors have shown that when haemophilia A patients with inhibitor are treated by regular infusion of high doses of FVIII, a treatment also called desensitisation or tolerance induction (see above), one of the biological consequences of such infusions is the elicitation of specific anti-idiotypic antibodies able to neutralise the inhibitor (Gilles J G et al. in (1996) J Clin Invest 97: 1382-1388). These findings suggest that the use of anti-idiotypic antibodies could represent a valuable approach for the control of FVIII inhibitory antibodies.
The human monoclonal antibody BO2C11 is a FVIII-specific IgG4kappa antibody derived from the natural repertoire of a patient with inhibitor (Jacquemin M G, et al. in (1998) Blood 92: 496-506). Ab BO2C11 recognises the C2 domain and inhibits the binding of FVIII to both vWF and phospholipids (PL). This antibody is representative of a major class of human inhibitory antibodies. Its mechanism of action is commonly encountered in patients with inhibitor and C2-specific antibodies are the most frequently observed inhibitory antibodies. Moreover, the exact binding site of Ab BO2C11 on the C2 domain has been deciphered through X-ray analysis of crystals made of the antibody Fab fragments and the C2 domain (Spiegel P. C. Jr. et al. (2001) Blood 98: 13-19).
Previous reports have described anti-idiotypic antibodies directed towards anti-FVIII antibodies. Lubahn and Reisner (1990, Proc Natl Acad Sci. USA 87: 8232-8236) partially purified human anti-FVIII antibodies by salt precipitation and chromatography from the plasma of a haemophilia A patient with inhibitor. These authors demonstrated that the purified IgG recognised the native FVIII heavy chain and its thrombin-digested 43 kDa chain. The preparation (SP8.4) was injected in mice in an attempt to obtain anti-idiotypic antibodies. Several clones were obtained, with one of them, Mab20-2H, inhibiting the anti-FVIII antibody binding to the heavy chain. In a functional assay, Mab20-2H did not modify the inhibitory activity of SP8.4 IgG fraction, even when added at high concentrations. Mab20-2H detected antibodies in 3.2% of the haemophilic inhibitor plasmas tested, but never neutralised the inhibitory activity. The authors concluded that Mab20-2H recognised a non-inhibitory anti-FVIII antibody that is directed toward the 43 kDa chain (A2 domain) of the FVIII molecule.
Another anti-idiotypic antibody (B6A2C1) was developed and described from the inventors' laboratory (Gilles J G et al in (1999) Blood 94, abstract 2048: 460a). This anti-idiotypic antibody is directed towards an anti-FVIII C1 domain inhibitor.